Use of cd31 peptides in the treatment of thrombotic and autoimmune disorders

ABSTRACT

The present invention stems from the finding that the extracellular domain of CD31 proteins present on blood leukocytes is shed and released in the circulation as a soluble form of CD31. The invention relates to peptides corresponding to fragments of CD31 that inhibit T-cell response, and to their use in the treatment of thrombotic disorders such as atherothrombosis and autoimmune disorders.

FIELD OF THE INVENTION

The present invention stems from the finding that the extracellulardomain of CD31 proteins present on blood leukocytes is shed and releasedin the circulation as a soluble form of CD31. The invention relates topeptides corresponding to fragments of CD31 that inhibit T-cellresponse, and to their use in the treatment of thrombotic and autoimmunedisorders.

BACKGROUND Thrombotic Disorders

In a healthy person, a homeostatic balance exists between procoagulant(clotting) forces and anticoagulant and fibrinolytic forces. Numerousgenetic, acquired, and environmental factors can tip the balance infavor of coagulation, leading to the pathologic formation of thrombi inveins (e.g. deep vein thrombosis), arteries (e.g. atherothrombosis,myocardial infarction, ischemic stroke), or cardiac chambers. Thrombican obstruct blood flow at the site of formation or detach and embolizeto block a distant blood vessel (e.g. pulmonary embolism, stroke).

Accumulating evidences show that atherothrombosis, a world-leadinglife-threatening disease, is linked to a defective immunoregulationdriving a pathologic activation of blood leukocytes and a destructiveinflammatory response within the vascular wall. Consequently, arestoration of immunoregulation at the blood-vessel interface wouldrepresent an innovative therapeutic option to fight atherothrombosis.

Autoimmune Disorders

In autoimmune disorders, the immune system produces antibodies to anendogenous antigen. Antibody-coated cells, like any similarly coatedforeign particle, activate the complement system, resulting in tissueinjury. Autoimmune disorders include systemic lupus erythematodes (SLE),rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory boweldisease (IBD), Graves' disease and diabetes mellitus.

Several mechanisms may account for the body's attack on itself.Autoantigens may become immunogenic because they are altered chemically,physically, or biologically. Certain chemicals couple with bodyproteins, making them immunogenic (as in contact dermatitis). Drugs canproduce several autoimmune reactions by binding covalently to serum ortissue proteins (see below). Photosensitivity exemplifies physicallyinduced autoallergy: Ultraviolet light alters skin protein, to which thepatient becomes allergic. In animal models, persistent infection with anRNA virus that combines with host tissues alters autoantigensbiologically, resulting in an autoallergic disorder resembling SLE.

Most human autoimmune diseases are specific antigen-driven T-celldiseases. T-cell clones responding to specific antigenic epitopes areresponsible for the initiation and/or the propagation of these diseases.Similarly, specific antigen-driven T-cell responses are responsible forthe rejection of organ allografts and the immune response to tumors.Activated T cells provide the “engine” for the chronic inflammation thatis associated with autoimmune diseases, organ graft rejection and tumorimmunity.

CD31 (PECAM-1)

Immune responses can be controlled by inhibitory immune receptors amongwhich CD31 (PECAM-1), which is expressed exclusively and constitutivelyon cells at the blood-vessel interface.

CD31 consists of a single chain molecule comprising six Ig-likeextracellular domains, a short transmembrane segment and a cytoplasmictail containing two ImmunoTyrosine-based Inhibitory Motif (ITIM)s. Thestructure of CD31 is shown in the table below.

Domain Position on SEQ ID No: 1 Signal peptide  1 to 27 Extracellulardomain  28 to 601 First Ig-like extracellular domain  34 to 121 SecondIg-like extracellular 145 to 233 domain Third Ig-like extracellulardomain 236 to 315 Fourth Ig-like extracellular 328 to 401 domain FifthIg-like extracellular domain 424 to 493 Sixth Ig-like extracellulardomain 499 to 591 Juxta-membrane domain 592 to 601 Transmembrane domain602 to 620 Cytoplasmic domain 621 to 738

The immunoregulatory properties of CD31 are supported by the fact thatCD31 signalling drives mutual repulsion of blood leukocytes andmodulates the balance between inhibitory and stimulatory signals of bothinnate and adaptive immune cells. Mechanical engagement of the distalIg-like extracellular domains of CD31 induces outside-in inhibitorysignalling triggered by the phosphorylation of its ITIMs, and therecruitment and activation of SH2-containing phosphatases.

Zehnder et al. (1995, Blood. 85(5):1282-8) identified a CD31 antibodythat inhibited the mixed lymphocyte reaction (MLR) in a specific anddose-dependent manner. They further found that a CD31 peptidecorresponding to the epitope of this antibody, i.e. to the 23membrane-proximal amino acids of CD31, strongly inhibited the MLR. Theyhypothesized that the 23 membrane-proximal amino acids of CD31constitutes a functionally important region, and that the CD31 peptideinterferes with lymphocyte activation by competing for binding epitopes.However, Zehnder et al. failed to teach whether CD31-mediated signalingis activated or inhibited by the CD31 peptide.

Chen et al. (1997, Blood. 89(4):1452-9) showed that this peptide delayedonset of graft-versus-host disease (GVHD) and increased long-termsurvival in a murine model of the disease. They hypothesized that theCD31 peptide inhibits a common pathway in T-cell activation. Again, Chenet al. failed to elucidate the role played by the CD31 peptide in T-cellactivation. In particular, these previous works did not assess theputative effect of the peptide on the CD31 signaling cascade and moreprecisely on the phosporylation state of the CD31 ITIMs.

By a yet unknown mechanism, CD31 is “lost” on certain circulatinglymphocytes. Its loss is observed upon lymphocyte activation and it hasbeen recently shown that the absence of lymphocyte CD31 signalling, inturn, heightens the pathologic immune responses involved in thedevelopment of atherothrombosis.

A soluble form of CD31, due to a variant transcript lacking thetransmembrane segment, has also been reported and therefore it iscurrently thought that the individual amount of circulating CD31 isgenetically determined. Consequently, a number of previous studies haveattempted to find a correlation between plasma levels of soluble CD31and the risk of atherothrombosis or other autoimmune diseases. However,independently of the specific genetic polymorphisms analyzed, datashowed a broad range of plasma CD31 values and the results of thesedifferent studies were contradicting.

There is therefore a need for better understanding the biologicalfunction of CD31. This would allow the provision of more efficienttherapeutics for the treatment of diseases linked with T-cellactivation.

DESCRIPTION OF THE INVENTION

It has surprisingly been found that the assumed loss of CD31 onactivated/memory T lymphocytes is actually incomplete and results fromshedding of CD31 between the 5^(th) and the 6^(th) extracellular Ig-likedomains. The shed extracellular domain of CD31 (further referred to as“shed CD31”) is then released into the circulation, where it is presenttogether with a soluble splice variant of CD31.

In addition, it has been shown that a high risk of atherothrombosis islinked with the increase in shed CD31 and decrease in splice variantCD31 in the circulation, and not with the total level of circulatingCD31.

The finding that CD31 is not lost on blood lymphocytes but only cleavedprovides a unique opportunity to rescue its physiologicalimmunoregulatory function by targeting the residual portion of themolecule. Specifically, the present invention provides peptidescorresponding to juxta-membrane amino acids of the ectodomain of CD31that are able to rescue the physiological immunoregulatory function ofCD31, even in patients having apparently lost CD31 from the surface oftheir circulating T lymphocytes.

It has been demonstrated that such peptides are capable of preventingdisease progression and aneurysm formation in a mouse model foratherosclerosis. Shorter and more stable peptides restricted to the last10 or 6 COOH-terminal amino acids of the known peptide of twenty-threeamino acids display superior in vitro immunosuppressive properties(lower ED and lower intra and inter-assay variability) than the knownpeptide. These amino acids correspond to a short extracellular fragmentcomprised between the membrane and the 6^(th) Ig-like domain of CD31.

The invention therefore provides peptides consisting of a fragmentcomprising the membrane juxta-proximal part of extracellular CD31 andpart of the sixth Ig-like domain and the use of such peptides in thetreatment of a thrombotic or an autoimmune as further described herein.

Such peptides have unique properties compared to soluble forms of CD31comprising all or most Ig-like domains of CD31. Indeed, such peptidesare highly homophilic since they have a Kd of 10⁻⁷ M, as assessed byBIAcore analysis. Hence they are able to engage CD31 signaling bybridging the membrane juxta-proximal part of extracellular CD31 thatremains expressed after its cleavage, via a strong homo-oligomerization.In contrast to this, alternatively spliced soluble CD31 lacks the first10 membrane juxta-proximal amino acids and shows weak homophilic bindingwith the 23-mer peptide (Kd of 17 μM, as assessed by BIAcore analysis).Furthermore, in vitro, only the peptides according to the invention arecapable of engaging the ITIM pathway downstream of the truncated isoformof CD31, and are thus capable of restoring CD31 signaling in Tlymphocytes having apparently lost CD31.

CD31 Peptides

It has been found that the six-amino-acid-long CD31 peptide of sequenceSEQ ID NO: 2 and that a ten-amino-acid-long CD31 peptide of sequence SEQID NO: 3 are capable of inhibiting T-cell activation. These two peptidescorrespond to fragments comprising the membrane juxta-proximal part ofextracellular CD31, which is adjacent to the sixth extracellular Ig-likedomain of CD31.

Therefore, the invention is directed to an isolated peptide comprisingor consisting of a fragment of CD31, wherein said fragment is selectedfrom the group consisting of:

-   -   a) a fragment of at least 3, 6 or 10 amino acids of the sequence        defined by amino acids 579 to 601 of SEQ ID NO: 1;    -   b) a fragment of at least 3, 6 or 10 amino acids of the sequence        corresponding to (a) in a non-human mammalian CD31; or    -   c) a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%        identical to (a);        with the proviso that said peptide of (c) does not consist of an        amino acid sequence of SEQ ID NO: 5 or 6.

As used herein, the term “peptide” has the meaning usually given in theart. More specifically, the dividing line between proteins and peptidesis usually set at a length of approximately 50 amino acids. Thus thepeptides according to the invention preferably have a length of at most50, 40, 35, 30, 25, 20, 15 or 10 amino acids.

As used herein, the term “fragment” of a reference sequence refers to achain of contiguous nucleotides or amino acids that is shorter than thereference sequence. More specifically, a fragment of the sequencedefined by amino acids 579 to 601 of SEQ ID NO: 1 is at most 22 aminoacids long. Said fragment may have a length of e.g. 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.Preferably, said fragment has a length within the range of 3 to 18, 3 to15, 3 to 10, 6 to 18, 6 to 15 or 6 to 10 amino acids.

The isolated peptide according to the invention may for example compriseor consist of a fragment having a sequence selected from the groupconsisting of SMRTSPRSSTLAVRVFLAPWKK (amino acids 2 to 23 of SEQ ID NO:5), MRTSPRSSTLAVRVFLAPWKK (amino acids 3 to 23 of SEQ ID NO: 5),RTSPRSSTLAVRVFLAPWKK (amino acids 4 to 23 of SEQ ID NO: 5),TSPRSSTLAVRVFLAPWKK (amino acids 5 to 23 of SEQ ID NO: 5),SPRSSTLAVRVFLAPWKK (amino acids 6 to 23 of SEQ ID NO: 5),PRSSTLAVRVFLAPWKK (amino acids 7 to 23 of SEQ ID NO: 5),RSSTLAVRVFLAPWKK (amino acids 8 to 23 of SEQ ID NO: 5), SSTLAVRVFLAPWKK(amino acids 9 to 23 of SEQ ID NO: 5), STLAVRVFLAPWKK (amino acids 10 to23 of SEQ ID NO: 5), TLAVRVFLAPWKK (amino acids 11 to 23 of SEQ ID NO:5), LAVRVFLAPWKK (amino acids 12 to 23 of SEQ ID NO: 5), AVRVFLAPWKK(amino acids 13 to 23 of SEQ ID NO: 5), VRVFLAPWKK (amino acids 14 to 23of SEQ ID NO: 5), RVFLAPWKK (amino acids 15 to 23 of SEQ ID NO: 5),VFLAPWKK (amino acids 16 to 23 of SEQ ID NO: 5), FLAPWKK (amino acids 17to 23 of SEQ ID NO: 5), LAPWKK (amino acids 18 to 23 of SEQ ID NO: 5),APWKK (amino acids 19 to 23 of SEQ ID NO: 5), PWKK (amino acids 20 to 23of SEQ ID NO: 5), WKK (amino acids 21 to 23 of SEQ ID NO: 5),HASSVPRSKILTVRVILAPWKK (amino acids 2 to 23 of SEQ ID NO: 6),ASSVPRSKILTVRVILAPWKK (amino acids 3 to 23 of SEQ ID NO: 6),SSVPRSKILTVRVILAPWKK (amino acids 4 to 23 of SEQ ID NO: 6),SVPRSKILTVRVILAPWKK (amino acids 5 to 23 of SEQ ID NO: 6),VPRSKILTVRVILAPWKK (amino acids 6 to 23 of SEQ ID NO: 6),PRSKILTVRVILAPWKK (amino acids 7 to 23 of SEQ ID NO: 6),RSKILTVRVILAPWKK (amino acids 8 to 23 of SEQ ID NO: 6), SKILTVRVILAPWKK(amino acids 9 to 23 of SEQ ID NO: 6), KILTVRVILAPWKK (amino acids 10 to23 of SEQ ID NO: 6), ILTVRVILAPWKK (amino acids 11 to 23 of SEQ ID NO:6), LTVRVILAPWKK (amino acids 12 to 23 of SEQ ID NO: 6), TVRVILAPWKK(amino acids 13 to 23 of SEQ ID NO: 6), VRVILAPWKK (amino acids 14 to 23of SEQ ID NO: 6), RVILAPWKK (amino acids 15 to 23 of SEQ ID NO: 6),VILAPWKK (amino acids 16 to 23 of SEQ ID NO: 6), ILAPWKK (amino acids 17to 23 of SEQ ID NO: 6), SSMRTSPRSSTLAVRVFLAPWK (amino acids 1 to 22 ofSEQ ID NO: 5), SSMRTSPRSSTLAVRVFLAPW (amino acids 1 to 21 of SEQ ID NO:5), SSMRTSPRSSTLAVRVFLAP (amino acids 1 to 20 of SEQ ID NO: 5),SSMRTSPRSSTLAVRVFLA (amino acids 1 to 19 of SEQ ID NO: 5),SSMRTSPRSSTLAVRVFL (amino acids 1 to 18 of SEQ ID NO: 5),SSMRTSPRSSTLAVRVF (amino acids 1 to 17 of SEQ ID NO: 5),SSMRTSPRSSTLAVRV (amino acids 1 to 16 of SEQ ID NO: 5), SSMRTSPRSSTLAVR(amino acids 1 to 15 of SEQ ID NO: 5), SSMRTSPRSSTLAV (amino acids 1 to14 of SEQ ID NO: 5), SSMRTSPRSSTLA (amino acids 1 to 13 of SEQ ID NO:5), SSMRTSPRSSTL (amino acids 1 to 12 of SEQ ID NO: 5), SSMRTSPRSST(amino acids 1 to 11 of SEQ ID NO: 5), SSMRTSPRSS (amino acids 1 to 10of SEQ ID NO: 5), SSMRTSPRS (amino acids 1 to 9 of SEQ ID NO: 5),SSMRTSPR (amino acids 1 to 8 of SEQ ID NO: 5), SSMRTSP (amino acids 1 to7 of SEQ ID NO: 5), SSMRTS (amino acids 1 to 6 of SEQ ID NO: 5), SSMRT(amino acids 1 to 5 of SEQ ID NO: 5), SSMR (amino acids 1 to 4 of SEQ IDNO: 5), SSM (amino acids 1 to 3 of SEQ ID NO: 5), NHASSVPRSKILTVRVILAPWK(amino acids 1 to 22 of SEQ ID NO: 6), NHASSVPRSKILTVRVILAPW (aminoacids 1 to 21 of SEQ ID NO: 6), NHASSVPRSKILTVRVILAP (amino acids 1 to20 of SEQ ID NO: 6), NHASSVPRSKILTVRVILA (amino acids 1 to 19 of SEQ IDNO: 6), NHASSVPRSKILTVRVIL (amino acids 1 to 18 of SEQ ID NO: 6),NHASSVPRSKILTVRVI (amino acids 1 to 17 of SEQ ID NO: 6),NHASSVPRSKILTVRV (amino acids 1 to 16 of SEQ ID NO: 6), NHASSVPRSKILTVR(amino acids 1 to 15 of SEQ ID NO: 6), NHASSVPRSKILTV (amino acids 1 to14 of SEQ ID NO: 6), NHASSVPRSKILT (amino acids 1 to 13 of SEQ ID NO:6), NHASSVPRSKIL (amino acids 1 to 12 of SEQ ID NO: 6), NHASSVPRSKI(amino acids 1 to 11 of SEQ ID NO: 6), NHASSVPRSK (amino acids 1 to 10of SEQ ID NO: 6), NHASSVPRS (amino acids 1 to 9 of SEQ ID NO: 6),NHASSVPR (amino acids 1 to 8 of SEQ ID NO: 6), NHASSVP (amino acids 1 to7 of SEQ ID NO: 6), NHASSV (amino acids 1 to 6 of SEQ ID NO: 6), NHASS(amino acids 1 to 5 of SEQ ID NO: 6), NHAS (amino acids 1 to 4 of SEQ IDNO: 6), NHA (amino acids 1 to 3 of SEQ ID NO: 6), SMRTSPRSSTLAVRVFLAPWK(amino acids 2 to 22 of SEQ ID NO: 5), MRTSPRSSTLAVRVFLAPW (amino acids3 to 21 of SEQ ID NO: 5), RTSPRSSTLAVRVFLAP (amino acids 4 to 20 of SEQID NO: 5), TSPRSSTLAVRVFLA (amino acids 5 to 19 of SEQ ID NO: 5),SPRSSTLAVRVFL (amino acids 6 to 18 of SEQ ID NO: 5), PRSSTLAVRVF (aminoacids 7 to 17 of SEQ ID NO: 5), RSSTLAVRV (amino acids 8 to 16 of SEQ IDNO: 5), SSTLAVR (amino acids 9 to 15 of SEQ ID NO: 5), STLAV (aminoacids 10 to 14 of SEQ ID NO: 5), TLA (amino acids 11 to 13 of SEQ ID NO:5), HASSVPRSKILTVRVILAPWK (amino acids 2 to 22 of SEQ ID NO: 6),ASSVPRSKILTVRVILAPW (amino acids 3 to 21 of SEQ ID NO: 6),SSVPRSKILTVRVILAP (amino acids 4 to 20 of SEQ ID NO: 6), SVPRSKILTVRVILA(amino acids 5 to 19 of SEQ ID NO: 6), VPRSKILTVRVIL (amino acids 6 to18 of SEQ ID NO: 6), PRSKILTVRVI (amino acids 7 to 17 of SEQ ID NO: 6),RSKILTVRV (amino acids 8 to 16 of SEQ ID NO: 6), SKILTVR (amino acids 9to 15 of SEQ ID NO: 6), KILTV (amino acids 10 to 14 of SEQ ID NO: 6) andILT (amino acids 11 to 13 of SEQ ID NO: 6).

In a preferred embodiment, said fragment corresponds to a juxta-membranefragment, i.e. to a fragment immediately adjacent to the transmembranedomain of CD31. In other words, said fragment preferably corresponds toa fragment including the C-terminal extremity of the sequence consistingof amino acids 579 to 601 of SEQ ID NO: 1 or of the correspondingsequence in a non-human mammalian CD31. For example, a peptideconsisting of a juxta-membrane fragment of 15 amino acids of thesequence defined by amino acids 579 to 601 of SEQ ID NO: 1 consists ofamino acids 587 to 601 of SEQ ID NO: 1.

In a specific embodiment, peptides comprising the transmembrane domainof CD31 or part thereof are excluded from the scope of the presentinvention.

The sequence of CD31 peptides according to the invention is preferablyderived from the sequence of human or murine CD31. However, the sequenceof CD31 may be derived from any non-human mammalian CD31 sequence. FIG.1 shows an alignment between the human, murine, bovine and pig CD31sequences. The sequence corresponding to the sequence defined by aminoacids 579 to 601 of SEQ ID NO: 1 is highlighted by a box. The skilled inthe art can easily identify the corresponding sequence in anothernon-human mammalian CD31 protein by performing a sequence alignment withthe sequences shown in FIG. 1.

In addition to the CD31 fragment, the peptide may optionally comprisesequences heterologous to CD31. These heterologous sequences may e.g.correspond to a carrier molecule such as the Keyhole Limpet Hemocyanin(KLH), bovine serum albumine (BSA), ovoalbumin (OVA), thyroglobulin(THY) or the multiple antigenic peptide (MAP).

In a preferred embodiment, the peptide comprises or consists of apeptide selected from the group consisting of:

-   -   the six-amino-acid-long peptide shown as SEQ ID NO: 2, which is        present both in human and murine CD31 sequences;    -   the ten-amino-acid-long peptide shown as SEQ ID NO: 3, the        sequence of which is derived from the mouse CD31 sequence; and    -   the ten-amino-acid-long peptide shown as SEQ ID NO: 4, the        sequence of which is derived from the human CD31 sequence.

In another preferred embodiment, the peptide comprises or consists of anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to any one of SEQ ID NOs: 2, 3 or 4.

CD31 peptides according to the invention have the biological activity ofexerting a dose-dependent inhibition of T-cell proliferation in vitroand/or of inhibiting the mixed-lymphocyte reaction (MLR). Theirbiological activity may for example be measured as described in Example3, Example 6 or in Zehnder et al. (1995, Blood. 85(5):1282-8).

The T-cell proliferation assay may comprise comparing the radioactivityincorporated into T-cells cultured either in the presence or in theabsence of the compound to be tested. This assay may for example beperformed as follows:

-   -   providing a multi-well plate comprising complete medium        supplemented with anti-CD3 antibodies;    -   supplementing the wells with increasing concentrations of the        compound to be tested;    -   plating peripheral blood mononuclear cells (e.g. of spleen        cells);    -   culturing the cells for about 72 hours;    -   adding (³H) thymidine and culturing the cells for about 16        hours;    -   measuring the radioactivity; and    -   comparing the radioactivity measured in the presence of the        compound to be tested with the radioactivity measured in the        absence of said compound, and/or in the presence of a reference        compound, and/or in the presence of a negative control.

Alternatively, the T-cell proliferation assay may comprise comparingexpression levels of the early activation marker CD69 in T-cellscultured either in the presence or in the absence of the compound to betested. This assay may for example be performed as follows:

-   -   providing purified CD4+ cells (e.g. purified from C57B16 mice);    -   stimulating said CD4+ cells by addition of anti-CD3 purified        antibodies and bone marrow derived dendritic cells;    -   culturing the cells for about 18 hours; and    -   analyzing said cells for the expression of the early activation        marker CD69, e.g. by flow cytometry; and    -   comparing CD69 expression in the presence of the compound to be        tested.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. Thus for instance, programs available in theWisconsin Sequence Analysis Package, version 9.1, for example theprograms BESTFIT and GAP, may be used to determine the % identitybetween two polynucleotides and the % identity and the % homologybetween two polypeptide sequences. BESTFIT uses the “local homology”algorithm of Smith and Waterman and finds the best single region ofsimilarity between two sequences. Other programs for determiningidentity and/or similarity between sequences are also known in the art,for instance the BLAST family of programs, accessible through the homepage of the NCBI at world wide web site ncbi.nim.nih.gov) and FASTA.

Peptides consisting of an amino acid sequence “at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical” to a reference sequence maycomprise mutations such as deletions, insertions and/or substitutionscompared to the reference sequence. In case of substitutions, thesubstitution preferably corresponds to a conservative substitution asindicated in the table below. In a preferred embodiment, the peptideconsisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to a reference sequence only differs from thereference sequence by conservative substitutions. In another preferredembodiment, the peptide consisting of an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencesequence corresponds to a naturally-occurring allelic variant of thereference sequence. In still another preferred embodiment, the peptideconsisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to a reference sequence corresponds to ahomologous sequence derived from another non-human mammalian speciesthan the reference sequence.

Conservative substitutions Type of Amino Acid Ala, Val, Leu, Ile, Met,Pro, Amino acids with aliphatic hydrophobic Phe, Trp side chains Ser,Tyr, Asn, Gln, Cys Amino acids with uncharged but polar side chains Asp,Glu Amino acids with acidic side chains Lys, Arg, His Amino acids withbasic side chains Gly Neutral side chain

In a preferred embodiment, the peptide according to the inventionconsists of any one of the sequences shown in the table below:

Swissprot SEQ accession ID species number position sequence NO: HumanP16284 592-601 VRVILAPWKK  4 Mouse Q08481 581-590 VRVFLAPWKK  3 RatQ3SWT0 580-589 VRVFLAPWKK 10 Pig Q95242 593-602 VRVYLAPWKK 11 BovinP51866 591-600 VRVYL-PLEK 12

CD31 peptides according to the invention may be prepared by anywell-known procedure in the art, such as solid phase synthesis, liquidphase synthesis or genetic engineering. As a solid phase synthesis, forexample, the amino acid corresponding to the C-terminus of the peptideto be synthesized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the C-terminus to the N-terminus, and one where the amino acidsbound to the resin or the protective group of the amino groups of thepeptides are released, the peptide chain is thus extended in thismanner. After synthesis of the desired peptide, it is subjected to thedeprotection reaction and cut out from the solid support.

The CD31 peptides of the invention may optionally comprise chemicalmodifications improving their stability and/or their biodisponibility.Such chemical modifications aim at obtaining peptides with increasedprotection of the peptides against enzymatic degradation in vivo, and/orincreased capacity to cross membrane barriers, thus increasing itshalf-life and maintaining or improving its biological activity. Anychemical modification known in the art can be employed according to thepresent invention. Such chemical modifications include but are notlimited to:

-   -   modifications to the N-terminal and/or C-terminal ends of the        peptides such as e.g. N-terminal acylation (preferably        acetylation) or desamination, or modification of the C-terminal        carboxyl group into an amide or an alcohol group;    -   modifications at the amide bond between two amino acids:        acylation (preferably acetylation) or alkylation (preferably        methylation) at the nitrogen atom or the alpha carbon of the        amide bond linking two amino acids;    -   modifications at the alpha carbon of the amide bond linking two        amino acids such as e.g. acylation (preferably acetylation) or        alkylation (preferably methylation) at the alpha carbon of the        amide bond linking two amino acids.    -   chirality changes such as e.g. replacement of one or more        naturally occurring amino acids (L enantiomer) with the        corresponding D-enantiomers;    -   retro-inversions in which one or more naturally-occurring amino        acids (L-enantiomer) are replaced with the corresponding        D-enantiomers, together with an inversion of the amino acid        chain (from the C-terminal end to the N-terminal end);    -   azapeptides, in which one or more alpha carbons are replaced        with nitrogen atoms; and/or    -   betapeptides, in which the amino group of one or more amino acid        is bonded to the β carbon rather than the α carbon.

By an “isolated” peptide, it is intended that the peptide is not presentwithin a living organism, e.g. within human body. However, the isolatedpeptide may be part of a composition or a kit. The isolated peptide ispreferably purified.

The compounds of the invention may be produced by any well-knownprocedure in the art, including chemical synthesis technologies andrecombinant technologies.

Examples of chemical synthesis technologies are solid phase synthesisand liquid phase synthesis. As a solid phase synthesis, for example, theamino acid corresponding to the C-terminus of the peptide to besynthesized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the C-terminus to the N-terminus, and one where the amino acidsbound to the resin or the protective group of the amino groups of thepeptides are released, the peptide chain is thus extended in thismanner. Solid phase synthesis methods are largely classified by the tBocmethod and the Fmoc method, depending on the type of protective groupused. Typically used protective groups include tBoe (t-butoxycarbonyl),Cl—Z (2-chlorobenzyloxycarbonyl), Br—Z (2-bromobenzyloyycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmcthoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl),Z (benzyloxycarbonyl) and Clz-Bzl (2,6-dichlorobenzyl) for the aminogroups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl)for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).After synthesis of the desired peptide, it is subjected to thede-protection reaction and cut out from the solid support. Such peptidecutting reaction may be carried with hydrogen fluoride ortri-fluoromethane sulfonic acid for the Boc method, and with TFA for theFmoc method.

Alternatively, the peptide may be synthesized using recombinanttechniques. In this case, a nucleic acid encoding a peptide according tothe invention (further referred to as “a nucleic acid according to theinvention”) is cloned into an expression vector. The nucleic acid of theinvention is preferably placed under the control of expression signals(e.g. a promoter, a terminator and/or an enhancer) allowing itsexpression. The expression vector is then transfected into a host cell(e.g. a human, CHO, mouse, monkey, fungal or bacterial host cell), andthe transfected host cell is cultivated under conditions suitable forthe expression of the peptide.

The method of producing the peptide may optionally comprise the steps ofpurifying said peptide, chemically modifying said peptide, and/orformulating said peptide into a pharmaceutical composition.

The invention also encompasses a method for identifying a peptidomimeticof a peptide according to the invention, said peptidomimetic being acandidate compound for the treatment and/or prevention of a thromboticor an autoimmune disorder, comprising the steps of:

-   -   a) providing a peptidomimetic; and    -   b) determining whether said peptidomimetic exerts:        -   i. a dose-dependent inhibition of T-cell proliferation in            vitro and/or of the mixed-lymphocyte reaction (MLR);        -   ii. an immunosuppressant activity; and/or        -   iii. an antiplatelet activity.            wherein, if said peptidomimetic exerts a dose-dependent            inhibition of T-cell proliferation in vitro and/or of the            mixed-lymphocyte reaction (MLR), an immunosuppressant            activity and/or an antiplatelet activity, said            peptidomimetic is a candidate compound for the treatment            and/or prevention of a thrombotic or an autoimmune disorder.

Step (a) of the above method may comprise designing and synthesizingsaid peptidomimetic.

Step (b) of the above method for identifying a peptidomimetic of apeptide according to the invention may comprise or consist of (i); (ii);(iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i) and (ii) and(iii).

Methods for determining whether said peptidomimetic exerts adose-dependent inhibition of T-cell proliferation in vitro and/or of themixed-lymphocyte reaction (MLR), an immunosuppressant activity and/or anantiplatelet activity are well known to the skilled in the art.

The determination whether said peptidomimetic exerts a dose-dependentinhibition of T-cell proliferation in vitro and/or of themixed-lymphocyte reaction (MLR) may for example be performed asdescribed in Example 3.

Immunosuppressant and antiplatelet activity can for example be theevaluated by analyzing the expression of activation surface markers(e.g. CD62P for platelets and/or CD69 for leukocytes) or of solubleactivation markers (e.g. TXA2 for platelets and/or IL-2 forLymphocytes).

As used herein, the term <<peptidomimetic>> refers to a compoundcontaining non-peptidic structural elements that mimics the biologicalaction of a CD31 peptide according to the invention. Methods fordesigning and synthesizing peptidomimetics of a given peptide arewell-known in the art and include e.g. those described in Ripka and Rich(Curr Opin Chem Biol. 1998; 2(4):441-52) and in Patch and Barron (CurrOpin Chem Biol. 2002; 6(6):872-7).

Use of CD31 Peptides for the Treatment of Thrombotic and AutoimmuneDisorders

It has been found that CD31 peptides corresponding to fragmentscomprising part of the sixth extracellular Ig-like domain of CD31 arecapable of activating CD31-mediated signaling, even in CD31⁻ (i.e.CD31^(shed)) T lymphocytes. In addition, such peptides are capable ofpreventing disease progression and aneurysm formation in a mouse modelfor atherosclerosis.

Therefore, the invention is directed to an isolated peptide comprisingor consisting of

a) amino acids 579 to 601 of SEQ ID NO: 1;

b) the amino acids corresponding to (a) in a non-human mammalian CD31;

c) a fragment of at least 6 amino acids of (a);

d) a fragment of at least 6 amino acids of (b); or

e) a sequence at least 80% identical to (a) or (c);

for use in activating CD31-mediated signaling. These peptides preferablyexert a dose-dependent inhibition of T-cell proliferation in vitro. Theactivation of CD31-mediated signaling may be an in vitro or an in vivoactivation.

As used throughout the present specification, the term “CD31-mediatedsignaling” refers to a signaling pathway in which CD31 is involved. Suchpathways are well known in the art and include those described e.g. inNewman and Newman (2003 Arterioscler Thromb Vasc Biol 23:953-964) and inNewton-Nash and Newman (1999. J Immunol 163:682-688).

The invention is further directed to an isolated peptide comprising orconsisting of:

a) amino acids 579 to 601 of SEQ ID NO: 1;

b) the amino acids corresponding to (a) in a non-human mammalian CD31;

c) a fragment of at least 6 amino acids of (a);

d) a fragment of at least 6 amino acids of (b); or

e) a sequence at least 80% identical to (a) or (c);

for use in the treatment of a thrombotic or an autoimmune disorder.These peptides preferably exert a dose-dependent inhibition of T-cellproliferation in vitro.

As used throughout the present specification, the term “thromboticdisorder” includes but is not limited to atherothrombosis,atherosclerosis, acute coronary syndrome, ischemic stroke, peripheralarterial disease and abdominal aortic aneurysm.

As used throughout the present specification, the term “autoimmunedisorder” includes but is not limited to rheumatoid arthritis (RA),multiple sclerosis (MS), inflammatory bowel disease (IBD), systemiclupus erythematodes (SLE), Graves' disease and diabetes mellitus.

In a preferred embodiment of the invention, said thrombotic orautoimmune disorder is associated with a loss of CD31⁺ T lymphocytesphenotype. Indeed, it has been surprisingly found that CD31 peptidesrestore CD31 signaling even in individuals with a CD31⁻ T lymphocytesphenotype. Therefore, in the context of the present invention, CD31peptides are preferably used to treat a subgroup of individuals and/orpatients having a CD31⁻ T lymphocytes phenotype.

As used herein, the term “CD31⁻ T lymphocytes phenotype” is usedinterchangeably with the term “CD31^(shed) T lymphocytes phenotype”.These terms refer to the phenotype of an individual having apparentlylost CD31 on its circulating T cells when conventional prior art methodsfor detecting CD31, e.g. such as those described in Stockinger et al.(Immunology, 1992, 75(1):53-8), Demeure et al. (Immunology, 1996,88(1):110-5), Caligiuri et al. (Arterioscler Thromb Vasc Biol, 2005,25(8):1659-64) or Caligiuri et al. (Arterioscler Thromb Vasc Biol, 2006,26(3):618-23) are used. In such methods, the antibody used for detectingCD31 binds to an epitope located on any one of the 1^(st) to the 5^(th)extracellular Ig-like domains.

Preferably, individuals having a CD31⁻ T lymphocytes phenotype have lostat least 50%, 60%, 65%, 70%, 75%, 80%, 90% or 95% of their circulatingCD4⁺/CD31⁺ T lymphocytes. In other words, at least 50%, 60%, 65%, 70%,75%, 80%, 90% or 95% of their circulating T lymphocytes are CD31^(shed)lymphocytes. Either the plasmatic rate or the cellular rate of CD31⁻ Tlymphocytes, compared to CD31⁺ T lymphocytes, may be measured.

The CD31 peptide used for activating CD31-mediated signaling and/ortreating a thrombotic or an autoimmune disorder may be any one of thepeptides described in the above paragraph entitled “CD31 peptides”.

Alternatively, the CD31 peptide used for activating CD31-mediatedsignaling and/or treating a thrombotic or an autoimmune disordercomprises or consists of:

-   -   a) amino acids 579 to 601 of SEQ ID NO: 1;    -   b) the amino acids corresponding to (a) in a non-human mammalian        CD31;    -   c) a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%        identical to (a) or (b);

The peptides used herein may have a length comprised between e.g.10-100, 15-80, 20-60, 25-50 and 20-40 amino acids. These peptidespreferably comprise or consist of fragments immediately adjacent to thetransmembrane domain of CD31. The peptide may for example correspond toa peptide consisting of an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5 or 6. The peptidepreferably consists of SEQ ID NO: 5 or 6. These peptides may optionallycomprise chemical modifications improving their stability and/or theirbiodisponibility.

The invention is also directed to a method of treating or preventing athrombotic or an autoimmune disorder comprising the step ofadministering an effective amount of a peptide as described herein, or anucleic coding therefore, to an individual in need thereof. Saidindividual in need thereof preferably suffers from or is at risk ofsuffering from a thrombotic or an autoimmune disorder. Most preferably,said individual has a CD31⁻ T lymphocytes phenotype.

By “effective amount”, is meant an amount sufficient to achieve aconcentration of peptide which is capable of preventing, treating orslowing down the disease to be treated. Such concentrations can beroutinely determined by those of skilled in the art. The amount of thecompound actually administered will typically be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like. It willalso be appreciated by those of stalled in the art that the dosage maybe dependent on the stability of the administered peptide.

The individuals to be treated in the frame of the invention arepreferably human individuals. However, the veterinary use of CD31peptides for treating other mammals is also contemplated by the presentinvention.

Pharmaceutical Compositions

The CD31 peptides described herein may be formulated into apharmaceutical composition. Thus the invention contemplates apharmaceutical composition comprising any one of the above CD31 peptidesand a physiologically acceptable carrier. Physiologically acceptablecarriers can be prepared by any method known by those skilled in theart.

Pharmaceutical compositions comprising at least one peptide of theinvention include all compositions wherein the peptide(s) are containedin an amount effective to achieve the intended purpose. In addition, thepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Suitable pharmaceutically acceptablevehicles are well known in the art and are described for example inRemington's Pharmaceutical Sciences (Mack Publishing Company, Easton,USA, 1985), which is a standard reference text in this field.Pharmaceutically acceptable vehicles can be routinely selected inaccordance with the mode of administration, solubility and stability ofthe peptides. For example, formulations for intravenous administrationmay include sterile aqueous solutions which may also contain buffers,diluents and other suitable additives. The use of biomaterials and otherpolymers for drug delivery, as well the different techniques and modelsto validate a specific mode of administration, are disclosed inliterature.

The peptides of the present invention may be administered by any meansthat achieve the intended purpose. For example, administration may beachieved by a number of different routes including, but not limited tosubcutaneous, intravenous, intradermal, intramuscular, intraperitoneal,intracerebral, intrathecal, intranasal, oral, rectal, transdermal,buccal, topical, local, inhalant or subcutaneous use.

Dosages to be administered depend on individual needs, on the desiredeffect and the chosen route of administration. It is understood that thedosage administered will be dependent upon the age, sex, health, andweight of the recipient, concurrent treatment, if any, frequency oftreatment, and the nature of the effect desired. The total dose requiredfor each treatment may be administered by multiple doses or in a singledose.

Depending on the intended route of delivery, the compounds may beformulated as liquid (e.g., solutions, suspensions), solid (e.g., pills,tablets, suppositories) or semisolid (e.g., creams, gels) forms.

In a preferred embodiment, the compositions are presented in unit dosageforms to facilitate accurate dosing. The term “unit dosage forms” refersto physically discrete units suitable as unitary dosages for humansubjects and other mammals, each unit containing a pre-determinedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. Typical unit dosage forms include pre-filled, pre-measuredampoules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions. In suchcompositions, the compound of the invention is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems.

The expression “physiologically acceptable” is meant to encompass anycarrier, which does not interfere with the effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which is administered. For example, for parenteraladministration, the above active ingredients may be formulated in unitdosage form for injection in vehicles such as saline, dextrose solution,serum albumin and Ringer's solution.

Besides the pharmaceutically acceptable carrier, the compositions of theinvention can also comprise minor amounts of additives, such asstabilizers, excipients, buffers and preservatives.

The invention also contemplates a pharmaceutical composition comprisinga nucleic acid encoding the peptide of the invention in the frame ofe.g. a treatment by gene therapy. In this case, the nucleic acid ispreferably present on a vector, on which the sequence coding for thepeptide is placed under the control of expression signals (e.g. apromoter, a terminator and/or an enhancer) allowing its expression. Thevector may for example correspond to a viral vector such as anadenoviral or a lentiviral vector.

The invention further provides kits comprising a pharmaceuticalcomposition comprising a CD31 peptide of the invention and instructionsregarding the mode of administration. These instructions may e.g.indicate the medical indication, and/or the route of administration,and/or the dosage, and/or the group of patients to be treated.

All references cited herein, including journal articles or abstracts,published or unpublished patent application, issued patents or any otherreferences, are entirely incorporated by reference herein, including alldata, tables, figures and text presented in the cited references.

Although having distinct meanings, the terms “comprising”, “having”,“containing’ and “consisting of” have been used interchangeablythroughout this specification and may be replaced with one another.

The invention will be further evaluated in view of the followingexamples and figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B shows an alignment between the sequences of human, mouse, pigand bovine CD31. The sequence defined by amino acids 579 to 601 of SEQID NO: 4 (human CD31) and the corresponding sequence in mouse, pig andbovine CD31 is highlighted by a box.

FIG. 2 shows a representative example of 10-color flow-cytometryanalysis of human peripheral blood cells from a healthy donor. Isotypecontrols of antibodies anti-CD31 dom1 and anti-CD31 dom6 are shown inthe insets. Lymphocytes, Monocytes and Granulocytes were gated withinthe FSC/SSC scatter. B (CD20 A700+) and T (CD3 PE-TR+) lymphocytes wereidentified and gated within the “Lymphocytes” and CD8+ (PerCP) and CD4+(APC) subpopulations were gated within T lymphocytes. CD8+ and CD4+ Tcells were further analyzed for the expression of HLA-DR and CD45RA andaccordingly subdivided in activated (1), memory (2) and naïve (3) cells.All leukocytes were positive for CD31 dom6. Lack of dom1 increased fromnaïve (3) to memory (2) to activated (1) T cells.

FIG. 3A-B shows that the apparent loss of CD31 on lymphocytes is due toits extracellular shedding. a. Solubilized cell membrane-bound CD31molecules were extracted from cultured Jurkat CD4+ T cells and coupledto fluorescent beads. The percentage of dom1-bead-bound molecules is <6%in resting conditions and >99% 5′ after TCR engagement. b. Most solubleCD31 in culture supernatant (□) of TCR-activated T cells and in humanplasma (▪) consists of a single truncated fragment comprising dom1-dom5and lacking dom6. Negligible levels of truncated CD31 lacking both dom5and dom6 could be detected only in plasma.

FIG. 4A-B shows that a peptide homotypic of the residual extracellularfragment on CD31^(shed)T induces CD31-ITIM phosphorylation. a.Proliferative response to TCR engagement of human peripheral bloodmononuclear cells in the presence of increasing doses of CD31 peptide551-574. *p<0.05 vs dose “0”. b. Flow cytometry assessment of 686ITIMphosphorylation on solubilized membrane-bound CD31 from cultured JurkatCD4+ T cells. Solubilized proteins were captured by E9-PECAM-1.2 (dom6)functional CBA beads and detection was carried out by anti-pY686 rabbitsera followed by AlexaFluor®488-anti-rabbit secondary antibody. Thehistogram shows the Median Fluorescent Intensity (MFI)±the % of thevariability coefficient (CV %) of Alexafluor®488 (pY686) over 2000E9-PECAM-1.2 acquired beads. Pervan=positive control (pervanadate);CD3/CD28=anti-CD3 and anti-CD28 antibodies (1 μg/ml each); peptide=CD31peptide 551-574 (100 μM).

FIG. 5A-D shows that CD31 homotypic peptide 551-574 inhibits T-cellresponses. a. BIAcore® analysis of mouse CD31 peptide 551-574 homophilicbinding at two-fold stepwise dilutions of the analyte (12.5, 25, 50 and100 μM). Data are normalised against the control channels and expressedas ΔRU (Resonance Units). b. TCR-induced intracellular calciummobilisation determined by flow cytometry in Fluo-3AM-loaded spleencells. Data are expressed as MFI (530/30 nm). Grey arrow=addition ofeither anti-CD3/CD28 antibodies and crosslinker alone (▪=control) orwith peptide 551-554 at 100 μM () or with anti-mouse CD31 antibodies(◯). c. Proliferation in response to TCR-stimulation in CD31+/+ (blackcolumns) and CD31−/− (white columns) spleen cells. A dose-dependentinhibition is observed in CD31+/+ cells while only the highest dose ofthe peptide affect proliferation of CD31−/− splenocytes. No effect wasobserved with 100 μM dose of the scramble peptide on CD31+/+ cells(crisscross column). *p<0.05 vs previous peptide dose. d.Immunosuppressive effect of the peptide in the DTH model. *p<0.01 vsscramble (crisscross column) and 10 μM dose of peptide 551-574. Data areexpressed as mean±SEM.

FIG. 6A-C shows that CD31 peptide biotherapy prevents acceleration ofatherosclerosis and aneurysm formation. a. Mean±SEM of Atheroscleroticlesion surface area in serial cross-sections of the aortic root at 200,400, 600, 800 μm from the appearance of the first cusp in control (▪)and peptide 551-574 (□) treated mice. *p<0.05 vs control, ANOVA,repeated measure. b. The presence of an abdominal aortic aneurysm (AAA)was macroscopically, blindly evaluated by A.G. and A. N. after carefuldissection of the adventitial tissue. An aneurysm was present in 8/10(Exp #1) or 6/8 (Exp #2) control mice as opposed to only 1/8peptide-treated mice in both experiments (p<0.001 by Chi squared test).The images below show an example of aneurysm (AAA; arrows) as comparedto the absence of aneurysm (no AAA) of the abdominal aorta. c. Flowcytometry analysis of the effect of the peptide (50 μM) on CD8+ T cell(% cytolysis) and macrophage (MMP-2/9 activity) functions. Datarepresent mean±SEM of cultures from 3-4 mous/group. *p<0.05 vs control.

FIG. 7 shows the clinical score of EAE activity (paralysis level).“pepREG” refers to the peptide of SEQ ID NO: 3. “pepSCRA” refers to thepeptide of SEQ ID NO: 14.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 corresponds to the sequence of human CD31.

SEQ ID NOs: 2, 3, 4, 10, 11 and 12 correspond to CD31 peptides accordingto the invention.

SEQ ID NOs: 5 and 6 correspond to CD31 peptides for use in the methodsaccording to the invention.

SEQ ID NO: 7 corresponds to the sequence of murine CD31.

SEQ ID NO: 8 corresponds to the sequence of bovine CD31.

SEQ ID NO: 9 corresponds to the sequence of pig CD31.

SEQ ID NOs: 13 and 14 correspond to scramble peptides used as controls.

EXAMPLES Example 1 Material and Methods

Assessment of CD31⁺ and CD31^(shed) Blood Leukocytes.

Ten-color flow cytometry was performed on peripheral blood leukocytesfrom 5 healthy individuals either in basal conditions or after overnightstimulation with soluble 1 μg/ml of purified anti-CD3 antibody (R&DSystems). Ten-color flow cytometry was performed after erythrocytehypotonic lysis (10 minutes at 37° C. 1:10 v:v in Tris 10 mM, NH₄CI 155mM, KHCO₃ 10 mM, pH 7.4) on heparinized peripheral blood leukocytes from5 healthy individuals, fixed in PBS/Formaldehyde 1%/FCS 1% for 4 minutesat 37° C. prior to processing. All experiments on human blood wereapproved by the International Ethical committee (see world wide web pageclinicaltrials.gov; Identifier: NCT00430820). Pelleted cells wereincubated for 30 minutes at room temperature and protected from lightwith a cocktail of fluorescent monoclonal antibodies directed to CD3(PE-Texas Red), CD4 (PE-Cy7), CD8 (PerCP), HLA-DR (APC-Cy7), CD45RA(Pacific Blue), and CD31 (WM59, PE) from BD Biosciences and anti-CD20(AlexaFluor® 700) and anti-CD31 (PECAM 1.2, FITC) from Invitrogen (1 μlof each). At least 50,000 events were acquired in the lymphocyte gateusing a BD LSRII® equipped with 3 lasers (405, 488 and 633 nm) andanalysed with BD DIVA® 6.0 software.

Subtractive Measurement of Soluble CD31.

To detect the splice variant and truncated CD31 in plasma and theculture supernatant, a cytokine bead array (CBA®, BD) has beencustomized. Three differently functional CBA beads (A9, D5 and E9) werecoupled with either one of the following purified monoclonal anti-CD31antibodies JC70A (domain 1, DAKO), MEM-05 (domain 5, Zymed) and PECAM1.2 (domain 6, Invitrogen). The coupled beads were then incubated withthe plasma of the same 5 healthy controls (FIG. 2) or the culturesupernatant and positive binding of circulating CD31 was detected by afourth anti-CD31 monoclonal antibody, WM-59 (domains 1-2) coupled to PE(BD). The concentration of plasma CD31 including at least domain 1(JC70A), or domains 1 to 5 (MEM-05) or all the extracellular domains 1to 6 of CD31 (PECAM 1.2) was determined by analysing the medianfluorescent intensity of the detecting antibody on ≧1000 gated beads onsamples and serial dilutions of the same standard (recombinant, fulllength extracellular CD31, R&D Systems). The standard curve was obtainedfor each of the beads using the same known concentrations of therecombinant CD31 in order to overcome any bias due to differences inbinding affinity of the diverse antibodies. The concentration in ng/mlof CD31 determined with PECAM 1.2 coupled beads (dom 1-6) was subtractedfrom the one obtained using MEM-05 coupled beads to obtain the amount ofcirculating CD31 lacking dom6 (dom 1-5). The latter was subtracted fromthe concentration of CD31 obtained using the JC70A-coupled beads tocalculate the value of soluble CD31 lacking both dom 5 and 6 butcontaining at least domains 1 and 2 (dom 1-2).

Assessment of CD31-ITIM Phosphorylation.

Log-phase Jurkat cells (10⁷ cells/condition) were either leftunstimulated (negative control) or incubated with pervanadate (positivecontrol) or stimulated with anti-CD3 and anti-CD28 antibodies (R&DSystems, 1 μg/ml each) in the presence or absence of peptide 551-574(100 μM), or incubated with the peptide alone during 20 minutes. Cellswere then lysed with 1 ml of RIPA buffer on ice for 30 minutes,ultracentrifuged and 16 μl of the supernatant was incubated with PECAM1.2-coated Functional E9 CBA® beads (BD) for 2 hours at roomtemperature. Beads were subsequently washed with CBA washing buffer andincubated with 2 μl of undiluted rabbit anti-CD31 phospho-tyrosine 686(pY686) sera followed by two washings and incubation withAlexaFluor®488-conjugated (Fab′)₂ fragments (1:100 in CBA whashingbuffer) of goat-anti-rabbit IgG (Invitrogen). The beads (2000/condition)were finally analysed by flow cytometry in the FITC channel (530/30 nm)and data are expressed as Median fluorescence intensity (MFI)±thepercentage of the coefficient of variability (% CV) calculated with theDIVA 6.0® software (BD). Duplicate lysate aliquots and serial dilutionsof recombinant CD31 were incubated with the PECAM 1.2-coated beads andthe amount of dom1+ cell-bound CD31 was revealed using anti-CD31WM59-R-PE (dom1) and PECAM 1.2-FITC (dom6) antibodies (data shown inFIG. 3 a).

Fluorescent Peptide Binding.

For visualisation of peptide binding to CD31⁺ and CD31^(shed) CD4⁺ Tcells, freshly purified peripheral blood leukocytes prepared as abovewere washed with a buffered solution containing 2 mM EDTA (to avoidendocytosis of the peptide) and incubated overnight at room temperaturein a dark humidified chamber with 50 μM FITC-labelled CD31 peptide551-574 and 1:10 dilution of fluorescent monoclonal anti-CD31 (PE) andanti-CD4 (APC) antibodies (BD Biosciences) in a poly-D-Lysine coatedIbidi® 8-well culture chamber (Biovalley). Cells were then washed twice,nuclei counterstained with DAPI and digital images of a 0.3 μmintracellular section were acquired on a Zeiss Axiovert M200 microscope(x63 immersion objective) equipped with the ApoTome® and a cooledmonochromatic digital camera (Zeiss).

Calcium Mobilisation Assay.

Spleen cells from C57Bl/6 mice were prepared as described in Caligiuriet al. (2005 Arterioscler Thromb Vasc Biol 25:1659-1664). Cells wereincubated with Fluo-3AM (Invitrogen, # F1242) as per the instructions ofthe manufacturer. Fluorescence of calcium-bound tracer was measured inthe FITC channel on an LSRII® cytometer (BD Biosciences) prior to andduring 60 seconds following the addition of hamster anti-mouse CD3/CD28monoclonal antibodies (40 μg/ml each) and rat/hamster compBead® (1:50)either alone or in the presence of rat anti-mouse CD31 antibody (clone390, 10 μg/ml) or in the presence of CD31 peptide 551-574 (100 μM).Negative controls included rat IgG isotype control and scramble peptide.Antibodies and compBeads® were from BD Biosciences.

Plasmon Surface Resonance.

Homophilic binding association and dissociation constants werecalculated by surface plasmon resonance (BIAcore® 2000, GE). In brief,peptide 551-574 was coated at 3400 resonance units (RU) on CM5 chipsaccording to the manufacturer's instructions. Soluble peptide 551-574(12.5, 25, 50 and 100 μM in 200 μl of 10 mM HEPES pH 7.4, 150 mM NaCl,0.005% Tween 20) was injected at 20 μl/min at 25° C., on thepeptide-coated channel and on an uncoated channel. Dissociation wasmonitored for 300 seconds. Association (kon) and dissociation (koff)constants were calculated using the BlAevaluation® 3.0 Software (GE).Injection of peptide 551-574 on a channel coated with the scramblepeptide yielded negligible signal.

Evaluation of Immunoregulation In Vitro.

CD8⁺ T cell-mediated cytolytic activity against allogeneic mouse aorticsmooth muscle cells and measurement of macrophage gelatinase (MMP-2/9)activity were performed as previously described for human cells inCaligiuri et al. (2006 Arterioscler Thromb Vasc Biol 26:618-623) usingkits and reagents from Invitrogen. Briefly, primary cultures of FVB/Nmouse aorta smooth muscle cells were labelled with the lipophylic tracerDIO (green) and co-cultured for 3 hours with CD8+ T cell-enriched spleencells from C57Bl/6 mice (n=3 scramble peptide and n=3 peptide 551-574,50 μM). Cytolysis was evaluated by intracellular accumulation ofpropidium iodide (PI). Cells were analysed by flow cytometry and the %of cytolysis was calculated by expressing the number of dead (PI+) cellsamong the target (DIO+) cells. Intracellular MMP-2/9 (gelatinase)activity was measured by flow cytometry in 7-day bone-marrow derivedmacrophages from C57Bl/6 mice (n=3 scramble peptide and n=3 peptide551-574, 50 μM) three hours after the incorporation of OregonGreen®gelatine (MFI). T-cell proliferation was performed using either humanperipheral blood mononuclear cells of spleen cells from C57Bl/6(CD31^(+/+)) and CD31^(−/−) mice (Charles River France) as previouslydescribed (Caligiuri et al. 2005 Arterioscler Thromb Vasc Biol25:1659-1664). Briefly, cells were plated in triplicates at 0.2×10⁶cells/well in a U bottom 96-well plate in complete medium (RPMI 1640, 1%pyruvate, 1% glutamine, 1% penicillin-streptomicyne-fungizone, 10%decomplemented fetal calf serum, all from Invitrogen) containing 1 μg/mlanti-mouse CD3/CD28 or 5 μg/ml anti-human CD3 antibodies (BD) asappropriate. CD31 (551-574) and scramble peptide at 25, 50 and 100 μMfinal concentration were deposited in the wells just before cellplating. Plated cells were cultured for 72 hours in 5% CO2 at 37° C.(³H) thymidine (0.5 μCi/well) was added for the last 16 hours andproliferation evaluated using a Tomtec harvester and analysis on aWallac micro beta counter. Data are expressed as mean±SEM of cpm intriplicates.

Evaluation of Immunoregulation In Vivo.

Delayed type hypersensitivity (DTH) suppression was evaluated asdescribed in the “Current Protocols in Immunology (2001) 4.0.1-4.0.2Unit 4.2”. Briefly, TNCB (2-chloro-1,3,5-trinitrobenzene, Fluka #79874)was dissolved in acetone/olive oil (1:1 v/v) at a concentration of 10mg/ml. BALB/c mice (n=6/group) were primed by painting the shavedregions of the abdomen a with a total 0.2 ml of the preparation(n=6/group). The experiment included 3 groups for peptide 551-574 (10,50, 100 μM) and 1 group treated with scramble peptide at 100 μM). Fivedays after priming, 10 μl of the TNCB-solvent mixture was painted on theright pinna, 30 minutes after subcutaneous (interscapolar)administration of the peptide 551-574 or the scramble peptide. Earthickness increases were calculated by subtracting the thickness of theright and the left pinna of each mouse (right-left/left×100), measuredat 24 h with a dial caliper (“Pocotest”, Kroeplin Längenmesstechnick).The measure was performed 5 times on each ear and averaged for furtheranalysis. The immunosuppressive effect of the peptide was calculated as% suppression=(1−ΔTE/ΔTS)×100, where ΔT=(ear thickness 24 hr afterelicitation)−(baseline ear thickness), E=sensitised animals, S=treatedanimals. Data are expressed as mean±SEM.

Detection of Atherosclerotic Lesion Size and Aneurysm Formation.

Male 28-week old apolipoprotein E^(−/−) mice (n=8-10 mice/group) fromour breeding facility were maintained on a regular chow diet and keptunder standard conditions. Acceleration of atherosclerosis and aneurysmformation was induced by subcutaneous angiotensin II (Sigma, #A9525)infusion (1 mg/kg/d) for 28 days using osmotic minipumps (Alzet, #2004)as previously described (Daugherty et al. J Clin Invest 105:1605-1612).All experiments were approved by our institutional Ethical committee.Atherosclerotic lesion size was measured as previously described(Caligiuri et al. 2005 Arterioscler Thromb Vasc Biol 25:1659-1664).These experiments were repeated twice with similar results.

Peptides.

All experiments on human material were carried out using the humanpeptide sequence while the mouse equivalent was used in all mouseexperiments. The sequences of the peptides are shown in the table below.

Human NH2 - NHASSVPRSKILTVRVILAPWKK -COOH SEQ ID NO: 6 MouseNH2 - SSMRTSPRSSTLAVRVFLAPWKK -COOH SEQ ID NO: 5 ScrambleNH2 - SMPAVRSRFSATSLVTLKSRWPK - SEQ ID COOH NO: 13

Example 2 The Apparent Loss of CD31 at the Surface of Blood Lymphocytesis Due to its Shedding Between the 5th and 6th Extracellular Ig-LikeDomains

In order to establish whether the loss CD31 was restricted to part orextended to the totality of its 6 extracellular Ig-like domains, amulticolor flow cytometry analysis of whole blood leukocytes from 5healthy donors using two different antibodies specifically recognizingthe membrane-distal and membrane-proximal Ig-like domains of themolecule was performed. To be able to discriminate between the differentleukocyte populations and assess their state of maturation andactivation, a panel of lineage markers as well as the expression ofCD45RA and HLA-DR were simultaneously used. While the expression ofCD31, as detected by a monoclonal antibody specific for the firstdomains of CD31 (clone WM-59, dom1-2) was recognized on naïve but not onactivated/memory blood T cells, all cells expressed themembrane-proximal extracellular fragment of the molecule detected byanother monoclonal antibody specific for the 6^(th) Ig-like domain ofCD31 (clone PECAM 1.2, dom6), irrespective of their state ofmaturation/activation (FIG. 2).

Flow cell cytometry showed that T-cell receptor (TCR) engagement inducesa shift of >80% of blood resting T cells from a CD31 dom1⁺/dom6⁺ to adom1⁻/dom6⁺ (CD31^(shed)) phenotype. Molecular analysis of the membraneproteins from cultured T-cell lysates demonstrated that >99% of the Tcell-bound CD31 molecules drop the distal portion containing dom1already 5′ minutes after TCR stimulation in vitro (FIG. 3 a). Analysisof the supernatant showed that, simultaneously, a single truncatedsoluble protein limited to the first 5 Ig-like domains of CD31,accumulates in the culture supernatant (FIG. 3 b). Furthermore, theanalysis of the plasma of the same healthy donors showed that major partof soluble CD31 in plasma was constituted of a truncated moleculecomprising Ig-like domains 1 to 5 and specifically lacking themembrane-proximal 6^(th) domain (FIG. 3 b) that always remains anchoredto the apparently CD31-negative (CD31 dom1⁻) lymphocytes both in vitroand in vivo. Only a minimal fraction of soluble CD31 contained all 6extracellular domains predicted in the previously reported (Goldbergeret al. J Biol Chem 269:17183-17191) variant spliced form both in culturesupernatant and in plasma (FIG. 3 b). No significant other cleavage ofthe molecule occurs upstream of the 5^(th) domain since the latter wasvirtually always present concomitantly with the first domain in thetruncated soluble CD31 proteins (FIG. 3 b).

Example 3 A Peptide Contained in the Residual Extracellular CD31Fragment on CD31^(shed) T Cells Enhances Phosphorylation of CD31-ITIM

A CD31 dom6-derived synthetic peptide corresponding to thejuxta-membrane 23 aminoacids (551-574) of the ectodomain of the humanmolecule binds both to CD31 dom1⁺ and to CD31 dom1⁻ (CD31^(shed)) CD4⁺ Tlymphocytes ex vivo. Importantly, the binding of this peptide on T cellshas functional consequences on immune cell control since it exerteddose-dependent inhibition of human peripheral blood T-cell proliferationin vitro (FIG. 4 a). To assess whether the inhibitory effect of thepeptide could be mediated by homophilic binding and engagement of theCD31 signaling, the level of phosphorylation of the CD31 ITIM tyrosineat position 686 (₆₈₆ITIM) in cultured T cells was evaluated. Stimulationof the TCR by anti-CD3 and anti-CD28 antibodies alone or the solepresence of the peptide induced a minor increase of CD31 pY686 (FIG. 4b) but concomitant TCR-stimulation in the presence of the peptideboosted the phosphorylation the CD31 ₆₈₆ITIM by a factor of >23 (FIG. 4b).

Example 4 CD31 Homotypic Peptide 551-574 Inhibits a Large Array ofCell-Mediated Immune Responses In Vitro and In Vivo

To further test our hypothesis, in vivo experiments were performed, andthe murine equivalent of CD31 (551-574) peptide was thereforesynthesized. Its properties were evaluated in vitro and in vivo. Itshomophilic interaction by surface plasmon resonance analysis was firstestablished (FIG. 5 a). The association and dissociation constants wereof 134±120 M⁻¹s⁻¹ and 1.58E-03±1.07E-03 s⁻¹, respectively and theaffinity at equilibrium was 17.8±9.7 μM, in agreement with previousmeasurement done on recombinant human CD31 homo-dimerization using othersystems. It was next assessed whether the mouse CD31 (551-574) peptidewas able to inhibit calcium mobilization in response to theco-engagement of the CD3 and of the co-stimulatory molecule CD28 inspleen lymphocytes. The data showed that homophilic targeting of thejuxta-membrane portion of CD31 whit this small peptide is as anefficient immunoregulatory strategy as it is the co-ligation of CD3 andCD28 with the distal portion of CD31 by cross-linking the moleculesusing specific monoclonal antibodies (FIG. 5 b). The engagement of theCD31 molecular pathway by this peptide could attain effectivesuppression of antigen-driven lymphocyte responses in vitro and in vivo.It was found that peptide 551-574 inhibits in a dose-dependent mannerthe proliferation of spleen cells in response to stimulation of the Tcell-receptor in vitro (FIG. 5 c). At low dose, the effect of thepeptide was exclusively due to its homophilic binding with CD31molecules since no effect was observed on spleen cells from CD31^(−/−)mice (FIG. 3 c). However, at high concentrations the peptide can alsobind to other (low affinity) heterophilic ligands as suggested by itseffect at 100 μM dose also on cells lacking CD31 (FIG. 5 c). Thescramble peptide, used at the highest dose, was ineffective (FIG. 5 c).Finally, the therapeutic immunosuppressive potential of this peptide invivo in the context of matched, histocompatible antigen-driven immuneresponses was evaluated using a model of delayed type ofhypersensitivity. Distal recall of an hapten-elicited specific immuneresponse was suppressed in a dose-dependent manner by peptide 551-574 inthis model (FIG. 5 d). Effective immunosuppression was achieved by asingle subcutaneous administration of 50 μM of the peptide 551-574 whilethe scramble peptide was ineffective (FIG. 5 d).

Example 5 CD31^(shed) Cell-Targeting Peptide Biotherapy Prevents DiseaseProgression and Aneurysm Formation in Atherosclerotic Mice

The model of angiotensin II infusion into aged apolipoprotein E−/− mice(25) was used, this model closely mimicking atherothrombosis in humans.Daily subcutaneous administration of 50 μM of the peptide prevented boththe acceleration of plaque growth in the aortic root (FIG. 6 a) and theformation of atherothrombotic abdominal aortic aneurysms (AAA) in thismodel (FIG. 6 b). in vitro, the CD31-derived peptide 551-574 inhibitedboth the activity of matrix degrading enzymes in bone-marrow derivedmacrophages and CD8⁺ T cell-dependent cytolysis of murine aortic smoothmuscle cells (FIG. 6 c).

Example 6 In Silico and In Vitro Validation of the 10 Amino Acid-LongPeptide Candidate Peptide “PepReg”

A shorter nested peptide corresponding to the ten COOH terminal aminoacids was tested. This peptide is shown as SEQ ID NO: 3 and referred toas “PepReg”. As shown in the table below, this peptide was expected tobe more stable than the CD31 551-574 peptide of 23 amino acids.

TABLE Characteristics of PepReg vs the  23aa CD31 peptide in silicoPepReg CD31 564- CD31 551-574 574 (10aa) (23aa) sequence VRVFLAPWKKSSMRTSPRSSTLAVRV (SEQ ID FLAPWKK NO: 3) (SEQ ID NO: 5) Number of 10 23amino acids Molecular weight 1243.5 2606.0 Theoretical pl 11.17 12.31Formula C62H98N16O11 C116H193N35O31S1 - (Asp, Glu) /+ 0/3 0/5 (Arg,Lys)charges Instability index 25.38 65.09 Estimated half- 100 hours1.9 hours life (mammalian reticulocytes, in vitro) Aliphatic index107.00 68.83

The immunosuppressive properties of PepReg (10aa) vs the 23 aa parentpeptide were evaluated in vitro. Negatively purified CD4+ cells fromC57Bl6 mice were stimulated by soluble anti-CD3 purified antibodies andbone marrow derived dendritic cells. Cells from triplicate wells wereanalyzed for the expression of the early activation marker CD69 after 18hours culture in complete RPMI medium supplemented with 10% fetal calfserum. Flow cytometry showed that PepReg was at least as efficient asthe 23aa peptide in suppressing T cell activation as determined by thepercentage of CD4 cells expressing CD69. Interestingly, the effect wasmore reproducible (smaller standard deviation) and was observed withlower doses (50 μg/ml vs 100 μg/ml) of PepReg (10aa) as compared to theparent (23aa) peptide. The suppression of T cell proliferation by PepRegwas analyzed by [H3] thymidin incorporation and persisted up to 7 daysof culture at 37° C. in 10% serum demonstrating that the data regardingthe stability of the peptide obtained in sifico were validated in vitro.

Example 7 In Vivo Validation of the 10 Amino Acid-Long Peptide CandidatePeptide “PepReg” in the EAE Model

Experimental Autoimmune Encephalomyelitis (EAE), also calledExperimental Allergic Encephalomyelitis, is an animal model of MultipleSclerosis.

Twelve-week old female C57BL/6J mice were immunized with 300 μg ofMOG35-55 peptide emulsified in Complete Freund's Adjuvant 1:1 by volumecontaining 800 μg of nonviable desiccated Mycobacterium tuberculosisH37RA. A final volume of 200 μl of the emulsion was injectedsubcutaneously at 4 sites (50 μl/site) over the flanks. In addition, 300ng of Pertussis toxin was injected intravenously (retro-orbital plexus)on the same day and 2 days later. Clinical signs of EAE ware assesseddaily by the following scoring system: 0, no signs; 1, hindlimbweakness; 2, hindlimb weakness and tail paralysis; 3, hindlimb and tailparalysis; 4, hindlimb and tail paralysis and forelimb weakness; 5,moribund state; and 6, death. The peak (waxing phase) occured around day21. In this C57BL/6J mouse model, there was no waning phase as assessedin our laboratory up to day 41.

The experiment was carried out with ten mice per group. The mice of eachgroup were treating with either of:

-   -   PBS;    -   PepReg (SEQ ID NO: 3); or    -   PepScra (SEQ ID NO: 14).

The dosing was of 50 μg of peptide per mice and per day (i.e. about 2mg/Kg per day). The peptide was administered by a subcutaneousinjection.

Disease protection was associated with reduced infiltration of IL17+ andIFNg+ T helper CD4+ cells and increased proportion of regulatoryCD25+/foxP3 CD4+ cells in the central nervous system of the mice.

As shown on FIG. 7, PepReg is capable of arresting disease developmentin the waxing phase (Day 15) and reducing disease extension in theplateau phase (Days 21 through to 35).

The scrambled peptide was also beneficial, although less than PepReg.This result suggests that the amino acid composition rather than thesequence per se is important for the beneficial effect. This result hasimportant implications for the development of peptidomimetics.

Example 8 Detection of Shed CD31 in Plasma from Patients Suffering fromAtherothrombosis and in Unaffected Individuals

The total amount of CD31, the amount of shed CD31 and the amount ofspliced CD31 has been measured both in eleven individuals suffering fromatherothrombosis and in twenty-three unaffected individuals.

The group “Atherothrombosis” comprised eleven individuals suffering fromchest pain even at rest and presenting an abnormal coronarography.

The group “No Atherothrombosis” comprised twenty-three individuals. Asub-analysis was carried out on the group “No Atherothrombosis”, whichwas found to comprise:

-   -   eight individuals presenting a normal coronarography and a        normal carotid echodoppler in spite of chest pain;    -   four individuals presenting a normal coronarography in spite of        chest pain, but in whom atherosclerosis was detected by carotid        echodoppler; and    -   eleven individuals suffering from chest pain only on effort and        presenting an abnormal coronarography (i.e. suffering from        coronary atherosclerosis without thrombosis).

The total amount of CD31, the amount of shed CD31 and the amount ofspliced CD31 was determined as follows.

1. The total amount (1 μl/test) of beads (E9, coupled with clone JC70A,DAKO) was transferred to a conical tube and centrifuged at 200 g for 5minutes. The supernatant was carefully discarded and replaced with sameamount of serum enhancement buffer (BD #51-9002150), and incubated atroom temperature for 15 minutes.

2. The fluorescently-labeled antibody antibody mix (PE-WM59; FITC-HC1/6;PB-PECAM1.2) was prepared, each at 1 μg/ml, 1 μl each/condition.

3. 1 tube precondition was prepared, each containing 3 μl of a standarddilution or a plasma sample. The reconstituted beads were centrifuged at200 g for 5 minutes, the supernatant was discarded and the serumenhancement buffer was replaced with the fluorescently-labeled antibodymix. 3 μl of this solution was distributed in each of the tubescontaining the standard dilution and samples, and the solution incubatedfor 1 hour at 4° C. in the dark.

4. 150 μl of Washing buffer (BD #51-9003797) were added to each tube,and the signal was acquired.

As shown in the table below, the percentage of shed CD31 was higher inindividuals suffering from atherothrombosis than in unaffectedindividuals, in spite of the fact that all individuals were sufferingfrom chest pain.

CD31 Plasma Level (ng/ml) total splice shed Atherothrombosis (n = 11)11.55 ± 0.7  −7.02 ± 2.69  18.57 ± 2.67 No Atherothrombosis 11.58 ± 0.495.26 ± 1.850  6.31 ± 1.85 (N = 23) T-test Prob >F 0.9756 0.0007 0.0006

Total CD31 amounts were similar in the four groups, while the amount ofshed CD31 and the amount of spliced CD31 were significantly different ineach paired group comparison. Shed CD31 was increased in individualswith abnormal coronarography, with highest values in those sufferingfrom atherothrombosis. Splice CD31 was still present in patientssuffering from atherosclerosis without atherothrombosis, while it wasalmost undetectable in patients suffering from atherothrombosis.

These results demonstrate that high levels of CD31 soluble splicevariants associated with low levels of shed CD31 indicates that thepatient suffers from non specific chest pain, eventually associated withcarotid plaques. A slight increase of shed CD31 levels associated withnormal or reduced levels of CD31 soluble splice variants indicates thatthe patient suffers from atherosclerosis. An important increase of shedCD31 levels associated with undetectable amounts of CD31 soluble splicevariants indicates that the patient suffers from atherothrombosis.

Example 9 Discussion of the Results

Dysimmune diseases are linked to lack of appropriate control of immuneresponses. Atherosclerosis and its complications are not only due tometabolic disturbances but are increasingly recognized as a dysimmunedisease and an important current issue is the identification ofinterventional tools able to restore immunoregulation. It has beenpreviously shown that atherothrombotic manifestations such as plaquerupture and thrombosis (Caligiuri et al. 2005 Arterioscler Thromb VascBiol 25:1659-1664) or aneurysm complication (Caligiuri et al. 2006Arterioscler Thromb Vasc Biol 26:618-623) are associated with asignificant reduction of CD31+ T lymphocytes in the peripheral blood. Inthese previous works, we documented that lack of CD31 signaling onlymphocytes elicited pro-atherothrombotic immune responses whilst thepresence of CD31 on T cells was able to inhibit them.

Here it is demonstrated that the assumed loss of the molecule onactivated/memory T lymphocytes is actually incomplete and results fromshedding of CD31 between the 5th and 6th extracellular Ig-like domains.CD31 shedding occurred immediately after cell activation on Tlymphocytes and was accompanied by the accumulation of the truncatedmolecule in the supernatant together with trace levels of the splicedvariant produced by the cells. This finding was unsuspected because allcommercially available tests to detect plasma CD31 use antibodiesdirected to CD31 domains 1 to 5, and therefore cannot discriminatebetween the spliced variant (containing all the 6 extracellular domains)and the truncated (domains 1 to 5) forms of CD31. The subtractiveimmunosorbent assay described herein is able to discriminate between thetwo forms of soluble CD31 and precisely quantify the proportion of eachof them in the plasma. This assay showed that the major part of plasmaCD31 comprises domains 1 to 5 but lacks the membrane-proximal 6thdomain, which remains anchored to blood CD31 dom1-lymphocytes.Therefore, it is proposed to refer to these lymphocytes as CD31shedrather that CD31 “negative” cells. Previous work in vitro had indicatedthat CD31 shedding at an unidentified position N-terminal from thetransmembrane segment of the molecule can occur in endothelial cellsundergoing apoptosis (Ilan et al. 2001. Faseb J 15:362-372). For thefirst time, it is shown herein that shedding is responsible for the CD31(incomplete) loss on blood lymphocytes and that the circulating CD31consists mainly of a truncated form derived from its cleavage betweenthe Ig-like domains 5 and 6, rather than of the secreted spliced variantform. Genetic polymorphisms for CD31 have been described, but thepredictive value of soluble CD31 levels was conflicting either inatherothrombosis or other dysimmune diseases. In fact, while the amountof the spliced form can be predicted by different genetic variants, theproportion of the form resulting from protein shedding is not determinedby CD31 gene polymorphism. It is proposed that the disparity between thedifferent studies was due to fact that circulating CD31 is a mixture ofthe genetic variant and of the truncated form and discrimination betweenthe two forms of CD31 was not possible. The subtractive method describedherein will allow the differentiation of the prognostic value determinedby genetic variants of CD31 independently of that linked to CD31shedding.

The fact that CD31 is not completely lost on blood lymphocytes offers aunique opportunity to rescue its physiological immunoregulatory functionby targeting the residual extracellular portion of the molecule. Indeed,it has been documented herein that this can be achieved by a homotypicpeptide-based therapy, both in vitro and in vivo. Homophilic binding ofthis peptide dramatically enhanced the phosphorylation the CD31 686ITIMand inhibited their TCR-induced proliferation. Induction of CD31 ITIMphosphorylation by antibody-mediated cross-linking of CD31 and CD3surface molecules was previously known to inhibit calcium mobilizationinduced by anti-CD3 antibodies in human T-cell lines. Remarkably, it hasbeen found that targeting the juxta-membrane portion of CD31 whit thesmall homotypic peptide is as an efficient immunoregulatory strategy asit is the co-ligation of CD3 and CD28 with the distal portion of CD31 bycross-linking the molecules with antibodies. A selective small syntheticpeptide strategy is obviously simpler and might also be safer than usinglarge proteins, such as monoclonal antibodies and cross-linkers, for thebiotherapy of immunological disorders (Isaacs 2007. Curr Opin Pharmacol7:418-425).

With this idea in mind, it was assessed whether the engagement of theCD31 molecular pathway by this peptide could attain effectivesuppression of antigen-driven lymphocyte responses in vitro and in vivoin the context of matched, histocompatible antigen-driven immuneresponses. Distal recall of an hapten-elicited specific immune responsewas suppressed in a dose-dependent manner by a single subcutaneousadministration of the peptide. A similar protective effect of a singlepeptide shot was also observed in experimental autoimmuneencephalomyelitis (a mouse model of multiple sclerosis) and lasted forup to 5 days.

Consequently, it was evaluated whether rescuing of CD31-mediatedimmunoregulation by the synthetic peptide 551-574 could be employed in abiotherapy to fight atherothrombosis since CD31-mediatedimmunoregulation is typically lost in this disease. It was chosen to usethe angiotensin-induced model of atherothrombosis because the abruptacceleration of atherosclerotic plaque growth and the development ofabdominal aortic aneurysms complicated by a thrombus in this model areproduced simply by excess bioavailability of a physiologicalpeptide—angiotensin II—and does not require the use of surgery, genetransfer or high fat diet, each of which could considerably bias theinterpretation of the results. The CD31-peptide prevented both theacceleration of plaque growth in the aortic root and the formation ofatherothrombotic abdominal aortic aneurysms in this model. Such adramatic protective effect was superior to any therapeutic molecule evertested and equivalent to that achieved by genetic manipulation.

Macrophages and lymphocytes represent the most important immune cellsinvolved in the development of atherothrombosis. The local function ofthese cells injure the cellular and extracellular components of thearterial layers resulting in either plaque rupture and luminalthrombosis, when occurring in the fibrous cap of atheroscleroticplaques, or aneurysm formation and eventually rupture, when happening inthe outer layers of the artery. Degradation of the extracellular matrixis essentially due to the activity of macrophage-derived matrixmetalloproteases while death of arterial smooth muscle cells isputatively caused by T cell-mediated cytolysis. Remarkably, CD31+ Tlymphocytes exert an important immunosuppressive function on both thesephenomena which are conversely aggravated by CD31shed T cells. Anaberrant reduction of CD31+ cells in patients with atherothrombosisunderlies the defective immunoregulation observed in the disease. Hereit is shown that the CD31-derived peptide 551-574 inhibits both theactivity of matrix degrading enzymes and T cell-dependent cytolysis ofthe arterial wall cells. The immunoregulation conveyed by this peptideis as efficient as that exerted by immunoregulatory CD31+ T cells andhence may counterweight the loss of the physiologic CD31-dependentimmunoregulation in human atherothrombosis.

This is the first time that a peptide-based biotherapy is envisaged tocorrect the defective immunoregulation characteristic ofatherothrombosis and prevent development of the disease in patients. Inaddition, such biotherapy may broaden over to other debilitatingdysimmune diseases. In particular, experimental studies have suggestedthat CD31-signalling might play a protective role in rheumatoidarthritis (Wong et al. 2005. J Clin Immunol 25:19-28), multiplesclerosis (Graesser et al. 2002. J Clin Invest 109:383-392) andnon-alchoolic fatty liver disease (Goel et al. 2007. Am J PhysiolGastrointest Liver Physiol 293:G1205-1214).

Example 10 Evaluation of Ninety Six Peptides According to the Invention

The ninety six peptides consisting of a fragment having a sequenceselected from the group consisting of:

-   -   amino acids 2 to 23, 3 to 23, 4 to 23, 5 to 23, 6 to 23, 7 to        23, 8 to 23, 9 to 23, 10 to 23, 11 to 23, 12 to 23, 13 to 23, 14        to 23, 15 to 23, 16 to 23, 17 to 23, 18 to 23, 19 to 23, 20 to        23 and 21 to 23 of SEQ ID NO: 5; and    -   amino acids 2 to 23, 3 to 23, 4 to 23, 5 to 23, 6 to 23, 7 to        23, 8 to 23, 9 to 23, 10 to 23, 11 to 23, 12 to 23, 13 to 23, 14        to 23, 15 to 23, 16 to 23 and 17 to 23 of SEQ ID NO: 6; and    -   amino acids 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to        17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to        10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4 and 1 to 3 of        SEQ ID NO: 5; and    -   amino acids 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to        17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to        10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4 and 1 to 3 of        SEQ ID NO: 6; and    -   amino acids 2 to 22, 3 to 21, 4 to 20, 5 to 19, 6 to 18, 7 to        17, 8 to 16, 9 to 15, 10 to 14 and 11 to 13 of SEQ ID NO: 5; and    -   amino acids 2 to 22, 3 to 21, 4 to 20, 5 to 19, 6 to 18, 7 to        17, 8 to 16, 9 to 15, 10 to 14 and 11 to 13 of SEQ ID NO: 6;        are tested for confirming that these peptides induce CD31-ITIM        phosphorylation. CD31-ITIM phosphorylation is assessed by        studying the effect of increasing doses of the peptides        according to the invention on CD31-ITIM phosphorylation of        cultured Jurkat cells stimulated with anti-CD3 and/or anti-CD28        antibodies, as described in Example 3.

It is confirmed that the above peptides inhibit T-cell proliferationusing the protocol described by Caligiuri et al. (2005 ArteriosclerThromb Vasc Biol 25:1659-1664).

It is confirmed that the peptides can achieve effectiveimmunosuppression using the protocol described in the Current Protocolsin Immunology (2001, 4.0.1-4.0.2 Unit 4.2).

The efficiency of the peptides for treating a thrombotic disorder isconfirmed in the model of angiotensin II infusion into agedapolipoprotein E−/− mice, which closely mimicks atherothrombosis inhumans.

The efficiency of the peptides for treating an autoimmune disorder isconfirmed in the Experimental Autoimmune Encephalomyelitis (EAE) model,which is an animal model of Multiple Sclerosis, and in a model forrheumatoid arthritis.

Example 11 Evaluation of the Peptides According to the Invention in anAnimal Model of the Rheumatoid Arthritis (RA)

It is confirmed that the peptides according to the invention (e.g.PepReg and/or the peptides described in Example 10) are capable ofarresting disease development and/or reducing disease extension in ananimal model of Rheumatoid arthritis (RA).

Rheumatoid arthritis (RA) is a chronic and systemic inflammatoryautoimmune disorder that causes the immune system to attack the joints.The disease is characterized by aggressive synovial hyperplasia (pannusformation) and inflammation (synovitis), which, if left untreated, leadto progressive destruction of joint cartilage and bone. The destructivelesions result from both immune responses and non-antigen-specificinnate inflammatory processes. Several studies have shown that CD31μlays a critical role in this disease since the disease onset andprogression is accelerated in its absence.

DBA/1 mice are used in this experiment. Induction of RA is initiated on12 week-old mice. On day 0, mice are immunized intradermally at the baseof the tail with 150 μg of bovine type II collagen (CII) emulsified withan equal volume of Freund's complete adjuvant containing 200 μg of H37RAMycobacterium tuberculosis. On day 21, mice are given a booster(intradermal injection of 150 μg of bovine CII in Freund's incompleteadjuvant). Simultaneously, mice receive an intravenous injection of 50μg of LPS. Mice are followed up for two months. Following immunization,the animals develop an autoimmune polyarthritis that is characterized bysevere cartilage and bone erosions. Mouse collagen-induced RA sharesseveral clinical, histopathological and immunological features withhuman RA.

The peptides according to the invention are administered following oneof the below treatment schemes.

-   -   Scheme 1: Preventive administration. Two doses (50 and 100        mg/kg) of the peptide is administered to the mice one day before        the CII immunization and than either daily, or twice a week, or        weekly, for the study period (2 months). A peptide with a        scrambled sequence (i.e. a peptide comprising the same amino        acids as the peptide according to the invention, but not the        same sequence) is administered to a control group of mice.        Treatment is pursued for one month.    -   Scheme 2: Curative administration. The peptide (50 and 100        mg/kg) and the scramble peptide are administered to mice after        the beginning of the symptoms, either daily, or twice a week or        weekly until the end of the study. Equivalent groups of mice are        kept in conventional housing facilities and are bled weekly from        a tail vein to monitor bleeding time and specific pathogen        antibody raise in sera (CDTA, Orleans).

Arthritis development is monitored by physical examination 3 times perweek. Inflammation in each of the 4 paws is graded on a scale of 0 to 3,and the scores for the 4 paws will be cumulated (yielding a maximumscore of 12 per mouse). The arthritis index is calculated by dividingthe total score in the experimental mice by the number of arthriticmice.

Lesions in the joints are also followed. Ankle joints of mice areexcised 6 weeks after immunization and fixed in 10% buffered formalin,decalcified in 10% EDTA, embedded in paraffin, sectioned, and stainedwith hematoxylin and eosin. The intensity of synovial hyperplasia,cellular inflammation, and pannus formation is evaluated, and arthritisis graded in a blinded manner on a scale of 0 to 4.

Immunohistochemistry is further used to track and to phenotypeinflammatory cells infiltrated in the joints.

The immunoregulation status is evaluated by measuring levels of serumIgG1 and IgG2a to CII. The measurement is performed by enzyme-linkedimmunosorbent assay (ELISA). The proliferation of T cells isolated fromdraining lymph nodes and the spleen is tested by the incorporation of ³Hthymidine in response to CII-loaded dendritic cells.

Cell populations in the lymphoid organs and in the synovia is analyzedby flow cytometry.

This experiment allows confirming that continuous administration of thepeptides according to the invention prevents onset of RA. The curativephase allows evaluating the therapeutic potential of the peptides fortreating RA in patients that do not respond to the current biologicals(i.e. 40% of the patients). Dose and frequency of the administrationsable to drive a regression of the inflammatory cells in the joints, andregression of the clinical score, are also determined.

1. An isolated peptide consisting of: a) a fragment of at least 6 aminoacids of the sequence defined by amino acids 579 to 601 of SEQ ID NO: 1;b) a fragment of at least 6 amino acids of the sequence corresponding to(a) in a non-human mammalian CD31; or c) a fragment consisting of asequence at least 80% identical to (a), wherein said peptide exerts adose-dependent inhibition of T-cell proliferation in vitro and does notconsist of an amino acid sequence of SEQ ID NO: 5 or
 6. 2. The peptideaccording to claim 1, wherein said peptide consists of: a) a fragment of6 to 15 amino acids of the sequence defined by amino acids 579 to 601 ofSEQ ID NO: 1; b) a fragment of 6 to 15 amino acids of the sequencecorresponding to (a) in a non-human mammalian CD31; or c) a fragmentconsisting of a sequence at least 80% identical to (a), wherein saidpeptide exerts a dose-dependent inhibition of T-cell proliferation invitro.
 3. The peptide according to claim 1, wherein said fragment is ajuxta-membrane fragment.
 4. The peptide according to claim 1, whereinsaid peptide comprises an amino acid sequence at least 80% identical toSEQ ID NO: 2, 3 or
 4. 5. The peptide according to claim 4, wherein saidpeptide comprises any one of SEQ ID NOs: 2, 3 or
 4. 6. The peptideaccording to claim 1, wherein said peptide comprises at least onechemical modification improving its stability and/or itsbiodisponibility.
 7. An isolated nucleic acid encoding a peptidecomprising a) a fragment of at least 6 amino acids of the sequencedefined by amino acids 579 to 601 of SEQ ID NO: 1; b) a fragment of atleast 6 amino acids of the sequence corresponding to (a) in a non-humanmammalian CD31; or c) a fragment consisting of a sequence at least 80%identical to (a), wherein said peptide exerts a dose-dependentinhibition of T-cell proliferation in vitro and does not consist of anamino acid sequence of SEQ ID NO: 5 or
 6. 8. A pharmaceuticalcomposition comprising one or more of a peptide comprising a) a fragmentof at least 6 amino acids of the sequence defined by amino acids 579 to601 of SEQ ID NO: 1; d) a fragment of at least 6 amino acids of thesequence corresponding to (a) in a non-human mammalian CD31; or e) afragment consisting of a sequence at least 80% identical to (a), whereinsaid peptide exerts a dose-dependent inhibition of T-cell proliferationin vitro and does not consist of an amino acid sequence of SEQ ID NO: 5or 6, and a nucleic acid encoding said peptide; and a physiologicallyacceptable carrier.
 9. A peptide comprising or consisting of: a) aminoacids 579 to 601 of SEQ ID NO: 1; b) the amino acids corresponding to(a) in a non-human mammalian CD31; c) a fragment of at least 6 aminoacids of (a); d) a fragment of at least 6 amino acids of (b); or e) asequence at least 80% identical to (a) or (c), wherein said peptideexerts a dose-dependent inhibition of T-cell proliferation in vitro; foruse in activating CD31-mediated signaling in vivo.
 10. A peptidecomprising or consisting of: a) amino acids 579 to 601 of SEQ ID NO: 1;b) the amino acids corresponding to (a) in a non-human mammalian CD31;c) a fragment of at least 6 amino acids of (a); d) a fragment of atleast 6 amino acids of (b); or e) a sequence at least 80% identical to(a) or (c), wherein said peptide exerts a dose-dependent inhibition ofT-cell proliferation in vitro; for use in the treatment of a thromboticor an autoimmune disorder.
 11. The peptide according to claim 9, whereinsaid peptide has a length of at most 30 amino acids.
 12. The peptideaccording to claim 9, for use in the treatment of individuals having aCD31⁻ T lymphocytes phenotype.
 13. The peptide according to claim 9,wherein said peptide comprises at least one chemical modificationimproving its stability and/or its biodisponibility.
 14. The peptideaccording to claim 9, wherein said peptide consists of: a) the peptideaccording to claim 1; b) a peptide comprising an amino acid sequence atleast 80% identical to SEQ ID NO: 5 or 6; or c) a peptide comprising anamino acid sequence of SEQ ID NO: 5 or
 6. 15. The peptide according toclaim 9, wherein said disorder is a thrombotic disorder selected fromthe group consisting of atherothrombosis, atherosclerosis, acutecoronary syndrome, ischemic stroke, peripheral arterial disease andabdominal aortic aneurysm.
 16. The peptide according claim 9, whereinsaid disorder is an autoimmune disorder selected from the groupconsisting of rheumatoid arthritis, multiple sclerosis, inflammatorybowel disease, systemic lupus erythematodes, Graves' disease anddiabetes mellitus.
 17. A method for identifying a peptidomimetic of apeptide according to any claim 1, being a candidate compound for thetreatment and/or prevention of a thrombotic or an autoimmune disorder,comprising the steps of: a) providing a peptidomimetic; and b)determining whether said peptidomimetic exerts a dose-dependentinhibition of T-cell proliferation in vitro and/or of themixed-lymphocyte reaction (MLR); wherein, if said peptidomimetic exertsa dose-dependent inhibition, said peptidomimetic is a candidate compoundfor the treatment and/or prevention of a thrombotic or an autoimmunedisorder.
 18. The method according to claim 17, wherein step (b) furthercomprises determining whether said peptidomimetic exerts animmunosuppressant and/or an antiplatelet activity.
 19. The peptideaccording to claim 10, wherein said peptide has a length of at most 30amino acids.
 20. The peptide according to claim 10, for use in thetreatment of individuals having a CD31⁻ T lymphocytes phenotype.
 21. Thepeptide according to claim 10, wherein said peptide comprises at leastone chemical modification improving its stability and/or itsbiodisponibility.
 22. The peptide according to claim 10, wherein saidpeptide consists of: a) the peptide according to claim 1; b) a peptidecomprising an amino acid sequence at least 80% identical to SEQ ID NO: 5or 6; or c) a peptide comprising an amino acid sequence of SEQ ID NO: 5or
 6. 23. The peptide according to claim 10, wherein said disorder is athrombotic disorder selected from the group consisting ofatherothrombosis, atherosclerosis, acute coronary syndrome, ischemicstroke, peripheral arterial disease and abdominal aortic aneurysm. 24.The peptide according claim 10, wherein said disorder is an autoimmunedisorder selected from the group consisting of rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease, systemic lupuserythematodes, Graves' disease and diabetes mellitus.